Over or insufficient angiogenesis often plays essential roles in the progression of many diseases, such as ischemic heart and limb, and tumor growth and metastasis. Angiogenesis is a process of formation of new blood vessels by sprouting from pre-existing ones. The long-term objective of this proposal is to elucidate the roles of angiopoietins in angiogenesis and determine how these roles are regulated. Angiopoietins are three closely related proteins that serve as ligands of Tie-2 receptor tyrosine kinase. Angiopoietin-Tie-2 pathway has been shown to play important role in embryonic angiogenesis. However, the roles of angiopoietins in tumor angiogenesis, which is essential for tumor growth and metastasis, is not well established, and the underlying cellular and molecular mechanism and the regulation of these roles are not well understood. We have demonstrated that Ang-1 binds to the extracellular matrix (ECM) and Ang-3 is retained to the cell surface via heparan sulfate proteoglycans (HSPGs), which modulate the function of Ang-1 or Ang-3, respectively. We hypothesize that Ang-1 and Ang-3 play important antagonistic roles in regulating endothelial cell behavior and angiogenesis in vivo and the pro-angiogenic role of Ang-1 is negatively regulated by its binding to the ECM and the anti-angiogenic role of Ang-3 is facilitated by its binding to cell surface HSPGs. The following specific aims are designed to test this hypotheses: To determine the molecular bases of the interactions between 1) Ang-1 and the ECM and 2) Ang-3 and the cell surface HSPGs; 3) to investigate how the binding of Ang-1 to the ECM and the binding of Ang-3 to the cell surface HSPGs affect their roles in regulating endothelial cell behavior and; 4) angiogenesis in vivo, We plan to accomplish the aims by using the established biochemical, molecular and cell biology approaches, the transfected cells, and the in vivo spontaneous pulmonary metastatic models of Lewis lung carcinoma cells. The completion of the proposed studies will generate new insights on the roles of Ang-1 and Ang-3 in endothelial cell biology and tumor angiogenesis and on how these functions of Ang-1 and -3 are regulated by the ECM or cell surface HSPGs, respectively. These results would contribute to our understanding of angiogenesis in general and help to develop potential novel and effective targets for therapies of the angiogenesis-related diseases, and are of high biologic and therapeutic relevance.